Phenylacetyl amino acid esters



Patented Mar. 8, 1949 PHENYLACETYL AMINO ACID ESTERS Otto K. Behrens,Indianapolis, Ind., assignor to Eli Lilly and Company, Indianapolis,Ind., a corporation of Indiana.

No Drawing. Original application August 24,

1945, Serial No. 612,524. Divided and this appllcation August 17, 1948,Serial No. 44,799

1 Claim.

This is a division of application Serial No.-

OOH

wherein R represents a member of the group consisting of hydrogen,methyl and ethyl, and R1 represents methyl or ethyl.

In the above formula, when R and R1 each represent methyl, the compoundis N-phenyl-- acetylvaline. When R represents ethyl and R1 representsmethyl, the compound is N-phenylacetylisoleucine. Additionally, when Rrepresents hydrogen and R1 represents methyl, the compound isN-phenylacetyl-a-amino-n-butyric acid. When R and R1 each representethyl, the compound is N-phenylacetyl-;9,B-diethylalanine. Furthermore,when R represents hydrogen and R1 represents ethyl, the compound isN-phenylacetylnor-valine.

The compounds of this invention may be employed in the form of theirfree acids and also in the form of their salts such as the sodium,potassium, calcium, magnesium, ammonium, and substituted ammonium salts,as well as in the form of their lower alkyl esters such as the methyl,ethyl, and propyl esters. Such salts and esters are to be regarded asbeing within the scope of this invention.

Broadly speaking, the compositions of this invention may be prepared byphenylacetylating the desired e-amino acid or its lower alkyl ester witha phenylacetyl halide such as, for example, phenylacetyl chloride. Thedesired phenylacetyloptically active base.

ation of the acid or ester may be accomplished Additionally, thephenylacetylated ester may The phenylacetylated ester, on the beprepared from the phenylacetylated acid by any suitable method ofesterification known to the art, such as reacting the phenylacetylatedacid in the presence of an alcohol and sulfuric acid.

Other methods of preparation of the compounds of this invention includethe reaction of a phenylacetyl halide with the amino acid or its loweralkyl ester in the presence of a tertiary-nitrogencontaining solventsuch as pyridine, quinoline, or triethylamine. Furthermore, thecompounds of this invention may be prepared by treatment of theaminoacid or ester with phenylacetic acid azide.

The compounds of this invention have an asymmetric carbon atom and aretherefore characterized by optically active forms. In accordance withthe presently preferred practice, reference to the form of the compoundsas used in the specification and claims is made on the basis of theconfigurational relationship of the isomers to an arbitrarily chosenstandard, namely, glyceric aldehyde, and is not based on observedoptical rotatory power. As used herein, the letters cl and l designatethe configurations of a compound relative to the arbitrary standard andirrespective of the sign of rotation, which latter is shown by thesymbol for the right-handed or dextr o, and for the left-handed orlevorotatory power. Thus, by way of example, l(+)-N-phenylacetylvalinerefers to the isomer which possesses the l-conflguration of the moleculeas determined by reference to the abovedescribed arbitrary standard anda or dextrorotatory power determinable by experimental observation.

Compounds of this invention which are characterized by the dorl-configuration of their molecules may be prepared by methods ofresolution well known to the art. Illustratively, chemical resolutionmay be employed, such as the preferential crystallization of a salt ofthe m-amino acid or phenylacetylated a-amino acid and an Furthermore,biological resolution may be employed, for example, the destruction ofone isomer of thee-amino acid by an amino acid oxidase.

As described and claimed in copending application Serial No. 612,526,filed August 24, 1945 (now Patent No. 2,440,356), compounds of thisinvention have been found to exhibit novel and unexpected utility in thepreparation of penicillin. By way of contrast, this utility is notpossessed by phenylacetylated amino acids in general. It is known, ofcourse, that penicillin may be produced, presumably as a metabolicproduct, when a Penicillium mold of the notatum-chrysogenum group isgrown in the presence of a nutrient medium and the penicillin soproduced may be subsequently isolated from the mixture of mold andnutrient medium.

As disclosed in said Patent No. 2,440,356, an accelerated production andan increased yield of penicillin may be obtained by incorporating arelatively small amount of a compound of the invention in the culturemedium of nutrient material in which the Penicillium mold is grown.

Illustratively, by carrying out the process of said Patent No. 2,440,356by employing, for example, dl-N-phenylacetylvaline in a submergedculture process the concentration of penicillin produced in certaintests under comparative conditions has been substantially increased.

For the purposes of the method described and claimed in said copendingapplication, the compounds of this inventionwhich are characterized bythe dl-form, which is a mixture of forms of the compounds exhibitingdand l-configurations of their molecules, function satisfactorily. Thel-forms of the compounds, however, are more efiicient in causing theincreased production of penicillin. The d-forms of the compounds whenemployed alone exhibit little if any activity in promoting theproduction of penicillin.

The following examples illustrate methods by which the compounds of thisinvention may be prepared:

Example 1 N-phenylacetylvaline represented by the following formula on,CH3- on OOHz-NH-fl coon may be prepared as follows:

To a solution of 200 g, of valine in 200 cc. of water and 450 cc. of 5 Nsodium hydroxide solution, there are added with stirring 293 cc. ofphenylacetyl chloride while maintaining the temperature below C. Theaddition is carried out over a period of two hours and when the bulk ofthe phenylacetyl chloride has been added, the reaction mixture is testedfrom time to time and kept alkaline by the addition of small quantitiesof sodium hydroxide solution. After all the phenylacetyl chloride hasbeen added, the solution is stirred for an additional minutes tocomplete the reaction. The reaction mixture is then filtered, acidifiedwith about 400 cc. of 6 N'hydrochloric acid solution and cooled to about0' C. whereupon the N-phenylacetylvaline crystallizes. The crystals arefiltered off, washed with cold water and dried. The N-phenylacetylvalineis suspended in one liter of percent ethylene dichloride-petroleum ethermixture, stirred well. filtered and washed with an additional 500 cc. ofethylene dichloride-petroleum ether mixture. N-phenylacetylvaline thusprepared has been found to melt at about 112-114" C.; and a micro- Dumasanalysis has shown the presence of 6.04

percent nitrogen as compared with a calculated value of 5.96 percent.

Example 2 N-phenylacetylvaline may also be prepared from phenylacetylchloride and valine in the following manner:

11.7 g. of valine are dissolved in 70 cc. of pyridine, and 16.9 g. ofphenylacetyl chloride are added in small portions with stirring whilemaintaining the temperature of the reaction below 40 C. The reactionmixture is allowed to stand atroom temperature for five hours after theaddition of. the. acid chloride has been completed. The reaction mixtureis then treated with about 25 cc. of water and evaporated almost todryness in vacuo. Another 25 cc. of water are then added and theevaporation repeated. The moist residue is suspended in about 25 cc. ofwater and acidified with hydrochloric acid whereuponN-phenylacetylvaline is completely precipitated from the solution. TheN-phenylacetylvaline is filtered off, dried, and purified by extractionwith a 50 percent ethylene dichloride-petroleum ether mixture.

Example 3 N-phenylacetylvaline additionally may be prepared from valineand phenylacetic acid azide in the following manner:

15 g. of phenylacetic acid hydrazide are dissolved and suspended in 150cc. of water containing 3.6 g. of hydrochloric acid. The aqueoussolution is layered with 50 cc. of ether and while maintained at about 0C. is treated with an aqueous solution of 6.9 g. of sodium nitrite.During the addition of the sodium nitrite the solution is stronglyagitated. The ethereal layer which contains the phenylacetic acid azideis dried with magnesium sulfate and added-with vigorous stirring to acold solution of 11.7 g. of valine dissolved in 50 cc. of 2 N sodiumhydroxide solution. The reaction mixture is stirred for about two hours.the ether layer separated and discarded, and the aqueous layer acidifiedwith hydrochloric acid. The N-phenylacetylvaline which precipitates isseparated by filtration, is dried, and is purified by extraction with a50 percent ethylene dichloride-petroleum ether mixture.

Example 4 l-(+)-N-phenylacetylvaline may be prepared in the same manneras the N-phenylacetylvaline of Example 1 except that l-valine is used asa starting material instead of dl-valine. l-(+) -N- phenylacetylvalinemelts at about 139-140" C. and has about the following optical rotationin 4 percent absolute ethanol solution:

[ lii"= +9.5

Example 5 l-(+)-N-phenylacetylvaline may also be prepared as follows, bychemical resolution by means of the brucine salt ofdl-N-phenylacetylvaline prepared according to Example 1. To a solutionof 23.5 g. of N-phenylacetylvaline in. 50 cc. of methanol is added asolution of 46.7 g. of

. brucine in 100 cc. of methanol and themixture allowed to evaporate atroom temperature. The

v residue is dissolved in about 450 cc. of hot. water and from the hotsolution upon cooling there separate colorless needles of the brucinesalt of l-(+) -N-phenylacetylvaline melting at about 105-108 C. Thissalt is recrystallized five times from water whereupon there is obtaineda purified salt melting at about 105 C. and having about the followingoptical rotation in 2 percent absolute ethanol solution:

14 g. of this purified brucine salt oil-(p-1 1- phenylacetylvaline aresuspended in cc. of

acoaeae water and treated with an excess of dilute sodium hydroxidesolution until the mixture becomes alkaline. The precipitated brucine isfiltered off and the flltrate acidified by treatment with hydrochloricacid, whereupon l-(+)-N-phenylacetylvaline precipitates. Thel-'(+)-N-phenylacetylvaline when recrystallized twice from dilutealcohol melts at about 139-140 C. and has about the following opticalrotation in 4 percent absolute ethanol solution:

Example 6 The methyl ester of N-phenylacetylisoleucine represented bythe following formula c 'OCHz-NH&H

may be prepared as follows:

18 g. of isoleucine methyl ester hydrochloride are dissolved in aminimum amount of water, the solution cooled to 0 C. and several volumesof ether added. To the solution are then added g. of 40 percent sodiumhydroxide solution with cooling and shaking. Solid anhydrous potassiumcarbonate is then added until the aqueous phase is converted to a pasteand separated. The aqueous paste is then extracted twice with ether, theether extracts added to the original extract, and the combined extractsdried with anhydrous magnesium sulfate. To the dry ether solution areadded 7 g. of phenylacetyl chloride in portions, and after this additionis completed, 7 g. of phenylacetyl chloride and 50 cc. of 10 per centsodium carbonate are added in small portions. The ether layer is thenwashed successively with dilute hydrochloric acid solution and sodiumbicarbonate solution and is dried over anhydrous magnesium sulfate. Thedried ether solution freed from the magnesium sulfate upon evaporationyields the methyl ester of N-phenyl acetylisoleucine.

Example 7 N-phenylacetyl-a-amino-n-butyric acid represented by thefollowing formula may be prepared from phenylacetyl chloride anda-aminobutyric acid by a method similar to that used for the preparationof N-phenylacetylvaline described in Example 1.N-phenylacetyl-uamino-n-butyric acid thus prepared has been found tomelt at about 124-126 C.; and a micro- Dumas analysis has shown thepresence of 6.44 percent nitrogen as compared with a calculated value of6.33 percent.

Example 8 N-phenylacetyl-fifi-diethy1alanine represented by thefollowing formula CzHs Czli's 0 Ce O l CH2CNH- H may be prepared asfollows:

To a mixture of 42 g. of ammonium chloride, 31.5 g. of sodium cyanideand cc. of methanol, 24.4 g. of a-ethylbutyr-aldehyde are added slowlywith stirring over a period of one half hour. Stirring is continued onehour longer and the mixture is then refluxed with stirring for fourhours. The solution is then cooled, 120 cc. of ether are added and themixture filtered. To the filtrate 120 cc. of concentrated hydrochloricacid are added over a period of one hour. During this addition hydrogencyanide is liberated. To the acidified mixture 120 cc. of water areadded and the mixture evaporated to remove all of the ether and most ofthe water. To the residue are added 30 cc. of concentrated hydrochloricacid and the solution refluxed for 10 hours. The resulting solutionafter evaporation to dryness in vacuo is treated with 30 cc. of waterand the evaporation in vacuo repeated. The residue is suspended in 10cc. of water, and cc. of 95 percent ethyl alcohol added, whereuponsodium chloride is precipitated and removed by filtration. 40 cc. ofaniline are then added to the filtrate and the mixture cooled to about 0C. for 10 or 12 hours. p,B-Diethylalanine precipitates from the solutionin crystalline form. The crystals are purified by washing them withabsolute alcohol and ether.

The 5,3-diethylalanine is converted intoN-phenylacetyl-ae-diethylalanine by treatment with phenylacetyl chlorideby substantially the same method used for the preparation ofN-phenylacetylvaline described in Example 1. N-phenylacetyl-8,B-diethylalanine thus prepared has been found to melt at about 98-100C.; and a micro-Dumas analysis has shown the presence of 5.6 percentnitrogen as compared with a calculated value of 5.3 percent.

Example 9 N-phenylacetylnor-valine represented by the formula ll IGomoawrmo 0 on may be prepared by reacting nor-valine with phenylacetylchloride in the presence of aqueous akali by the method used for thepreparation of N-phenylacetylvaline described in Example 1.

N-phenylacetylnor-valine has been found to melt at about 136-138 C.; anda micro-Dumas analysis has shown the presence of 5.95 percent nitrogenascompared with the calculated value of 5.96 percent.

Example 10 N-phenylacetylisoleucine represented by the formula C2115 CHOOH may be prepared by reacting isoleucine with phenylacetyl chloride inthe presence of aqueous alkali by the method used for the preparation ofN-phenylacetylvaline described in Example 1.

N-phenylacetylisoleucine has been found to melt at about 108-109 C.; anda micro-Dumas analysis has shown the presence of 5.45 percent nitrogenas compared with a calculated value of 5.62 percent.

laim 7 I 8 I c The methyl ester 0! N-phenylacetylisoleucine REFERENCESCITED represented by the formula The following references are of recordin the cg 01H; file of this patent:

L E 5 UNITED STATES PATENTS Q 3 Number Name Date 600cm 2,440,356 BehrensAug. 24, 1945 and characterized by the 1- configuration of its molecule.1

O'I'IO K. BEHRENS.

